Extraction of purine nucleotides from biologic substances



Patented Feb. 18, 1947 EXTRACTION OF PURINE NUCLEOTIDES FROM BIOLOGICSUBSTANCES Louis Laufer and David R. Schwarz, New York, N. Y., assignorsto Schwarz Laboratories, Inc., New York, N. Y., a corporation of NewYork No Drawing. Application September 15, 1943,

Serial No. 502,502

16 Claims. 1

Our present invention is concerned primarily with processes for therecovery of'valuable constituents from yeast and from other biologicsubstances that are rich in nucleic acid or in purine nucleotides assuch.

The invention constitutes an improvement upon that of the priorapplication, Serial No. 454,- 838, of Louis Laufer and Jesse Charney,filed August 14, 1942, in effecting considerable economy by enhancingand expediting the yield and saving chemical reagents.

In carrying out the present invention the nucleotides are dissolved byalkaline hydrolysis, and where the substance treated is yeast or othernucleic acid bearing substance, such nucleic acid is at the same timehydrolyzed to degrade it into its four nucleotide components, allwithout the need for first isolating the nucleic acid, as such, from thecell complex.

In this operation undesirable constituents, including protein and gummymaterial unavoidably pass into solution with the nucleotides. Thepresent invention provides a means by which such proteins and gummysubstances can readily be removed from such nucleotide solution, thusrendering the solution sufficiently pure for convenient furthertreatment.

According to the invention such removal of protein and gummy ingredientsis effected by seiective precipitation thereof and the nucleotides arerecovered from the final filtrate for subsequent separation therefrom ofthe pentose, if desired.

The resultant slurry may be filtered to recover therefrom as aby-product the insoluble materials including yeast cell walls, but inpractice it is preferred to treat the slurry as such Without firstremoving such materials therefrom.

The protein ingredients will precipitate in an acid medium, the gummymaterials in an alkaline medium and the precipitation of theseconstitu-.

ents is therefore effected successively in either order.

In a preferred procedure the protein material is removed first from theslurry, followed by removal of the gum. To this end the mass after theinitial treatment with alkali is adjusted with mineral acid, preferablysulphuric acid, to substantially the isoelectric point of the proteins,and

not previously removed and a brilliant filtrate is obtained. Theprecipitate may be used as a high protein feed adjunct or may beprocessed for other constituents.

The gums are then precipitated from the filtrate by the addition of analkali until the optimum point for such precipitation is reached and theprecipitated gums are then removed by mechanical separation as byfiltration or centrifugation.

The resultant clear filtrate includes in solution the various purinenucleotides and other ingredients, but the present invention is notconcerned with the latter which are inert to the subsequent treatment.

From this point on the treatment may follow closely the teaching of theprior application above-identified for precipitating the purinenucleotides as the cuprous salt thereof and for splitting off thepentose therefrom. By the present invention certain improvements havebeen made in some aspects of the recovery and treatment of the cuprouspurine nucleotides and these will be set forth hereinafter.

In the alternative procedure, the gummy substances are precipitated andremoved before the precipitation and removal of the proteins. The gumsare precipitated from the original unneutralized hydrolysate, which is arelatively strong alkaline medium, by the addition of copper sulphateand from the filtrate the protein is precipitated and removedsubstantially as in the preferred procedure.

The invention has been above set forth in its broad and general aspectsand various embodiments in detail will now be described.

In a preferred process one kilogram of pressed yeast, either brewers orbakers yeast, is treated with 2 to 3 percent by weight of caustic sodain 50 percent solution. The mixture is heated to between and degrees C.,and maintained at that temperature for about one hour with occasionalstirring. In this operation the mixture becomes fluid and the nucleicacid becomes freed from its protein combination and is also hydrolyzedinto its four nucleotide components which pass into solution togetherwith protein and gummy constituents of the yeast.

The mass is now diluted with two litres of water and the pH of themixture is adjusted to the pH range between 4.5 and 4.7 by the additionof concentrated sulphuric acid and thereupon boiled for from 15 to 20minutes. The pH specified being substantially the isoelectric point ofmost of the yeast proteins, these pass out of solution.

The boiling helps to denature and coagulate the proteins so that whenthe solution has been cooled to room temperature the proteins will havesettled out substantially completely. Preferably by filtration theproteins are removed together with those insoluble components. of theyeast including the cell walls which were separated in the originalalkali treatment. A brilliant filtrate results.

To the filtrate there is added sufificient sodium hydroxide to reach theoptimum point for precipitating the gums. The alkali used, preferablysodium hydroxide, would be about 0.2 per cent by weight for a strain ofbrewers yeast, of the gen.- eral classification known as Saccharomycescerevisiae, but, for other strains of yeast, would be elsewhere in therange between 0.2 and 0.5 per cent by weight. The precipitated gums areremoved by either filtration or centrifugation.

For precipitating the purine nucleotides the clarified filtrate isadjusted to pH 5.5 for the foregoing strain of brewers yeast, but forother strains of yeast that adjustment would be elsewhere in the rangebetween 5.5 and 8.5. Thereupon the solution is raised to the boilingpoint and 52 to 1% grams of crystalline copper sulphate are added. Thesecrystals promptly pass into solution. While the boiling continues thereis then added to the solution 10.5 to 21 grams of sodium bi-sulphite.

For best yield the amount of copper sulphate and sodium bi-sulphiteadded should be more or lessv proportional to the concentration ofnucleotides in the strainof yeast employed. The foregoing quantities arebest for the above identified strain of brewers yeast.

The pH of the solution after said nucleotide precipitating agents havebeen added should be between 2.9 and 3.1. In that pH range a maximum, infact a substantially quantitative precipitation of cuprous purinenucleotides occurs. The cuprous purine nucleotide precipitate is thenpromptly removed from the solution through a filter press or by acentrifuge.

The precipitate is washed several times with water to remove smallamounts of impurities. It is then treated substantially as set forth inthe prior application above-identified. Briefly stated, the cuprouspurine nucleotides are hydrolyzed in sulphuric acid, the resultantcuprous purines are filtered ofi, the filtrate is neutralized withbarium hydroxide, the resultant barium sulphate, barium phosphate andcopper phosphate are filtered ofi; the filtrate is concentrated invacuum, filtered, treated with activated carbon, again filtered,concentrated, transferred to diatomaceous earthactivated carbon mixture,dried in vacuum and extracted with ether-alcohol mixture. The resultantyield is substantially pure d-ribose. It varies from 1.5 to 3.0 gramsper kilogram of pressed brewers or bakers yeast, depending on the strainof yeast employed.

In the foregoing process the treatment of pressed yeast has been setforth, which is assumed to have a solid content of 30 to 35 per cent.The process could, however, be applied to liquid yeast which is assumedto have a solid content of only to per cent by using enough caustic inthe initial hydrolyzation to bring the liquid yeast to the same state ofalkalinity as set forth above for the pressed yeast.

In the alternative procedure, the gums are precipitated prior to theprecipitation of the proteins. That process also can be applied toyeast, either in pressed or liquid form. V For the treat-.

ment of liquid yeast, there is added to three kilograms thereof, enough50 per cent caustic solution to make the entire mass 2 to 3 per centcaustic by weight. The slurry is held at to degrees C. for one hour andthen cooled. Copper sulphate solution is then added, which serves toprecipitate the gums, even in the relatively strong alkaline medium inwhich, in the absence of such copper sulphate, they would remain insolution. A 5 per cent copper sulphate solution is preferred which byvolume should be between one fourth and one third of that of thesolution or slurry to which it is added. In practice 875 ml. of suchcopper solution has been found to give best results.

After removal of the precipitated gums, as for instance, by filtration,the protein is precipitated out by the procedure above outlined and thenucleotides are precipitated and the pentose split off therefrom also inthe manner above set forth. According to the procedure last set forth,the final yield of pure crystalline d-ribose is 1.9 to 2.0 grams perkilogram of pressed yeast, depending upon the strain of yeast used.

The procedure in which pressed yeast is used and the proteins areprecipitated prior to precipitation of the gums is preferred because, asappears from the foregoing, it involves considerably less cost forchemicals, in fact not much more than half what is required in thealternative procedure using liquid yeast. Moreover, in the preferredprocess, in which the proteins are precipitated first, the separation ofsolid from liquid is mechanically more satisfactory and less of thedesired nucleotide material is retained by the residual cake.Furthermore, the protein byproducts are uncontaminated by copper.

While the invention has been described and has its preferred applicationin the treatment of pressed yeast as perhaps one of the richest sourcesof nucleic acid, it is also applicable to the treatment of other nucleicacid bearing substances, and also of substances that contain nucleotidesas such, for instance to moulds, bacteria and blood cells.

As many changes could be made in the above process and many apparentlywidely different embodiments of this invention could be made withoutdeparting from the scope of the claims, it is intended that all mattercontained in the above description shall be interpreted as illustrativeand not in a limiting sense.

Having thus described our invention, what we claim as new and desire tosecure by Letters Patent of the United States is:

l. The process of extracting purine nucleotides from naturally occurringnucleic acid bearing biologic substance, comprising the heating of suchsubstances in the presence of aqueous solution of caustic for hydrolysisof the nucleic acid until the nucleotide constituents are freed and passinto solution, together with gum and protein constituents of thesubstance and thereupon precipitating out and removing the latterconstituents and finally precipitating the nucleotides from thesolution.

2. The process of extracting purine nucleotides from naturally occurringnucleic acid bearing biologic substance, comprising the addition to suchsubstance of aqueous caustic solution, the application of heat theretountil the nucleic acid has been freed from the substance and has beenhydrolyzed into its nucleotide components which pass into solution, theremoval from the solution by precipitation of both the gummy and theprotein constituents thereof and the separation of the purinenucleotides from the residual filtrate.

3. The process of extracting purine nucleotides from yeast, comprisingtreating such yeast with 2 to 3 per cent by Weight of sodium hydroxidein aqueous solution, maintaining the mass for in the order of one hourat a temperature in the order of 60 to 65 degrees (3., therebyliberating the nucleic acid from its protein combination and alsohydrolyzing it into its nucleotide componehts which pass into solution,while insoluble yeast constituents including cell walls remain insuspension, thereupon removing from the solution by precipitation boththe gummy and the protein constituents, and separating the nucleotidesfrom the residual filtrate.

4. The process of claim 3 in which the insoluble yeast constituents suchas cell walls are removed from the slurry with the precipitate ofprotein constituents brought down in an acid medium and prior to theremoval of the gummy constituents.

5. The process recited in claim 3 in which the insoluble yeastconstituents such as cell walls are removed from the slurry with theprecipitate of gummy constituents brought down by the addition of coppersulphate and prior to removal of the protein constituents.

6. The process recited in claim 3 in which the insoluble yeastconstituents such as cell walls are filtered out of the slurrry beforeprecipitating out the protein and ummy substances.

7. The process of extracting purine nucleotides from naturally occurringnucleic acid bearing biologic substance, comprising the heating of suchsubstances in the presence of aqueous solution of caustic until thenucleic acid has been hydrolyzed to said nucleotide constituents and thelatter pass into solution to ether with gummy and protein constituentsof the substance, followed by adjustment in the alkalinity or pH of thesolution to a range in which one of the two classes of constituentsconsisting of gummy material and proteins is precipitated out and afterseparation of such precipitate adjusting the alkalinity or pH to therange for precipitating out the remaining class of such constituents andafter removal of the latter precipitate separating out the nucleotidesfrom the residual filtrate.

8. The combination recited in claim 7 in which the protein constituentsare precipitated by the addition of mineral acid to adjust the pH tosubstantially the isoelectric point of such proteins at pH in the rangebetween 4.5 and 4.7 and boiling the mixture until such precipitation issubstantially complete and after filtering out the precipitate, addingcaustic to adjust the alkalinity to the range for optimum precipitationof the gums.

9. The combination recited in claim 7 in which the protein constituentsare precipitated by the addition of sulphuric acid to adjust the pH tosubstantially the isoelectric point of such proteins and boiling themixture until such precipitation is substantially complete and afterremoving the precipitate by mechanical procedure, adding caustic in therange between 0.2 and 0.5 per cent by weight to the filtrate, andremoving the precipitated gums by mechanical procedure.

10. The process recited in claim 7 in which the gummy constituents areprecipitated by the addition of copper sulphate to the alkaline solutionof nucleotides, gummy substances and proteins, and after filtering outsuch precipitate the filtrate is adjusted in pH to substantially theisoelectric point of the proteins and boiled until the protein matter isprecipitated.

11. The process recited in claim 7 in which the gummy constituents areprecipitated by the addition of 5 per cent copper sulphate solution involume between one fourth and one third that of the alkaline solution ofnucleotides, gummy substances and proteins and after filtering out suchprecipitate the filtrate is adjusted with sulphuric acid to a pH in therange of 4.5 to liland boiled for about fifteen minutes and then cooledto room temperature and the proteins thereby coagulated andprecipitated.

12. The combination recited in claim 7 as applied to the extraction ofpurine nucleotides from yeast in which the solution of nucleotides,gummy substances and proteins is filtered prior to the removal of gummysubstances and protein for the withdrawal of insoluble materialincluding the yeast cell walls.

13. The combination recited in claim 7 as applied to the extraction ofpurine nucleotides from yeast, in which the removal of insolublematerials including the yeast cell walls separated in the originalalkali treatment is effected after the precipitation of one of the groupof protein and gum substances and prior to the separation of the other.

14. The combination recited in claim '7 in which the purine nucleotidesare precipitated as the cuprous salts thereof.

15. The combination recited in claim 7 in which the purine nucleotidesare separated out by first adjusting the filtrate to the pH range of 5.5to 8.5, adding copper sulphate and sodium bisulphite and boiling, andthereupon mechanically removing the resulting precipitate ofsubstantially pure cuprous purine nucleotides.

16. The combination recited in claim 7 in which the purine nucleotidesare precipitated as the cuprous salts thereof by first adjusting thefiltrate to the pH range of 5.5 to 8.5, adding copper sulphate andsodium bisulphite until the pH of the solution has been reduced to therange of 2.9 to 3.1, boiling, and thereupon filtering out the resultingprecipitate of substantially pure cuprous purine nucleotides.

LOUIS LAUFER. DAVID R. SCHWARZ.

REFERENCES CITED The following references are of record in the file ofthis patent:

Nucleic Acids, Levene (ACS Monograph No. 56), pp. 221-4, 229-300.

